This revised proposal (DK47308-01A2) entitled Downstream elements in IRS-1 signaling: p55/PIK focuses on a novel protein discovered in our laboratory by cDNA expression screening with a [32/P]IRS-1 probe. IRS-1 is an essential component for insulin signaling that becomes phosphorylated on multiple tyrosine residues during activation of the insulin receptor kinase, as well as the receptors for IGF-1, IL-4 and growth hormone. We suspect that important insulin signals are engaged when the phosphorylated tyrosine residues associate with high affinity to cellular signaling proteins which contain Src homology-2 domains (SH2-proteins). The p55/PIK is a newly discovered SH2-protein which shares characteristics with p85, a principle regulatory element linking phosphatidylinositol-3 kinase to IRS-1 and other growth factor receptors. A full understanding of the IRS- 1 signaling system and the elements it interacts with is scientifically and clinically important because diabetes is a contemporary health problem that affects about 2% of the world population and over 14 million Americans. Whereas 10% of these individuals suffer from an absolute lack of insulin (IDDM), most are diabetic because their cells do not respond fully to normal or elevated amounts of circulating insulin (NIDDM). A molecular basis for insulin signaling will provide new therapeutic opportunities for NIDDM patients and improved strategies for the tight control of individuals with IDDM. This proposal introduces a new element in the insulin signaling system called p55/PIK, that appears to link the insulin receptor/IRS-1 to the PI 3-kinase and possibly other downstream signaling molecules. Based on growing literature pointing to the important of PI 3-kinase signaling, and glucose uptake in particular, p55/PIK is expected to play an important role in the cellular insulin response. This proposal will establish over the next five years the role of p55/PIK in the insulin signaling cascade and explore the role of p55/PIK in the regulation of glucose metabolism, neuronal development and apoptosis. The following specific aims are proposed: 1. The expression and function of p55/PIK will be determined in tissues from rats and mice under normal conditions and during fasting, obesity and insulin resistance. 2. The function of p55/PIK in cultured P19 neuronal cells will be investigated. 3. Structure/function analysis of p55/PIK will be conducted by site- directed mutagenesis and overexpression in cultured cells, including CHO, P19 neurons and 3T3-L1 adipocytes. 4. The gene for p55/PIK will be disrupted in mice, and the effect of on glucose homeostatis will be evaluated in the resulting animals.